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Abstract

Colorectal cancer is the second and third most leading cancer in Europe and the US, respectively. One of the main risk factors of this malignancy of the large bowel has been associated to western-style diets rich in high saturated fat and meat and low in fiber. During the last years, it has been reported that a regular uptake of fruits and vegetables may reduce the incidence of colorectal cancer. One of the most consumed fruits in western diet patterns are apples. Several epidemiological and case-control studies have indicated that usual consumption of one or more apples a day may reduce the risk for colon cancer. But so far, the possible preventive effects of apple juice have not been completely investigated. Our previous examinations showed that polyphenolic extracts from apple juice are biologically active in terms of radical scavenging, influence on drug metabolism as well as anti-inflammatory and anti-hormonal potential in vitro. Moreover, we used the colorectal chemoprevention Apc Min/+ mouse model to study the chemopreventive effect of apple juices. This strain is highly susceptible to spontaneous intestinal adenoma formation. The results showed that cloudy apple juice and polyphenolic apple juice extract had a significant impact diminishing the tumor development in the small intestine of Apc Min/+ mice. However, the possible mechanism remained to be understood. In the present study, the possible molecular mechanism was investigated in a second animal experiment. Animals were treated with water (as a control), cloudy apple juice (CAJ), placebo juice (apple juice without polyphenols), 0.2% B-PAE (procyanidin-rich apple extract) and a combination between cloudy apple juice and 0.2% B-PAE. Both cloudy apple juice and B-PAE intervention significantly reduced adenoma development by 24% and 34%, respectively. These findings indicated polyphenols as the main chemopreventive compounds in this model. On the other hand, placebo juice and cloudy apple juice plus B-PAE treatments, inhibited intestinal adenomas in a nonsignificant manner by 7% and 17%, respectively. Moreover, placebo juice intervention enhanced small adenomas in the whole small intestine. In the liver, cloudy apple juice- and cloudy apple juice plus B-PAE-treated animals resulted in an induction of Phase II enzymes (GST, QR, and TrxR). Further investigations identified a high concentration of polyphenol metabolites in the urine of animals treated with cloudy apple juice and cloudy apple juice plus B-PAE. This indicated that polyphenol metabolites may reach the liver after intestinal absorption. However, due to an overnight fasting prior to dissection, the protein content of liver cells of mice in the water and B-PAE intervention groups was disminished. Several enzymatic activies such as CYP1A1, QR and TrxR were decreased as a result of this fasting effect. In contrast, placebo juice, CAJ and CAJ + B-PAE maintained hepatic protein contents due to the carbohydrate uptake from these drinks. The possible mechanism of the development of adenomas was investigated using protein array technology. Proteomic analysis revealed that fasting effects increased phosphorylated ERK and the target iNOS in intestinal adenomas. Similarly, Cyclin D1 was reduced but only in the middle parts of the small intestine. B-PAE treatments reduced fasting associated effects by decreasing p-ERK, iNOS, and Cyclin D1. Since only very low concentrations of metabolites of polyphenols were found in urine and in analogy to other comparable compounds, a direct passage into the intestine or hepato-biliary excretion can be assumed. Therefore these results might be attributed to procyanidins acting locally. CAJ and B-PAE treatments did not affect inflammation markers such as nitric oxide content in urine or PGE2 in serum. In vivo levels of apple polyphenols were not high enough to efficiently inhibit these inflammation markers. Cyclin D1 was strongly reduced in the distal parts of the small intestine by CAJ and CAJ + B-PAE. As well as in a non-significant manner, beta-catenin was reduced in a similar way. Overall, apple polyphenols from CAJ and B-PAE reduced adenoma development in the Apc Min/+ mouse model. This chemopreventive potential was dependent on the intestinal bioavailability. Control animals (12 h fasted) resulted in an increase of Erk phosphorylation and of iNOS expression. B-PAE-treated animals suppressed these fasting-associated effects compared to water controls as well as in the groups that had access to high-calorie apple juice.